The Canine Papillomavirus and Gamma HPV E7 Proteins Use an Alternative Domain to Bind and Destabilize the Retinoblastoma Protein

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Abstract
The high-risk HPV E6 and E7 proteins cooperate to immortalize primary human cervical cells and the E7 protein can independently transform fibroblasts in vitro, primarily due to its ability to associate with and degrade the retinoblastoma tumor suppressor protein, pRb. The binding of E7 to pRb is mediated by a conserved Leu-X-Cys-X-Glu (LXCXE) motif in the conserved region 2 (CR2) of E7 and this domain is both necessary and sufficient for E7/pRb association. In the current study, we report that the E7 protein of the malignancy-associated canine papillomavirus type 2 encodes an E7 protein that has serine substituted for cysteine in the LXCXE motif. In HPV, this substitution in E7 abrogates pRb binding and degradation. However, despite variation at this critical site, the canine papillomavirus E7 protein still bound and degraded pRb. Even complete deletion of the LXSXE domain of canine E7 failed to interfere with binding to pRb in vitro and in vivo. Rather, the dominant binding site for pRb mapped to the C-terminal domain of canine E7. Finally, while the CR1 and CR2 domains of HPV E7 are sufficient for degradation of pRb, the C-terminal region of canine E7 was also required for pRb degradation. Screening of HPV genome sequences revealed that the LXSXE motif of the canine E7 protein was also present in the gamma HPVs and we demonstrate that the gamma HPV-4 E7 protein also binds pRb in a similar way. It appears, therefore, that the type 2 canine PV and gamma-type HPVs not only share similar properties with respect to tissue specificity and association with immunosuppression, but also the mechanism by which their E7 proteins interact with pRb. Human papillomaviruses (HPVs) are estimated to cause the most common sexually transmitted infection in the world, and these infections are recognized as the major cause of cervical cancer. One of the papillomavirus oncoproteins, E7, plays a major role in both the viral life cycle and progression to cancer. In cells E7 associates and inactivates pRb, a tumor suppressor protein. For the vast majority of papillomaviruses, E7 binds to pRb using a small amino acid sequence, LXCXE. However, we have now identified a papillomavirus E7 protein that lacks the LXCXE domain yet still binds and degrades pRb. This E7 protein, derived from a carcinogenic canine virus, uses its C-terminal domain to bind pRb. In addition, we discovered that a family of papillomaviruses, the gamma type HPVs, also lacks the LXCXE domain and binds pRb using a similar mechanism.